Use of fak inhibitor in preparation of drug for treating tumors having nras mutation

ABSTRACT

A use of a FAK inhibitor in the preparation of a drug for preventing and/or treating tumors having an NRAS mutation. A method for treating tumors that have experienced an NRAS mutation, which comprises administering an effective dose of a FAK inhibitor to an individual. A FAK inhibitor for treating tumors having an NRAS mutation. The FAK inhibitor is BI853520, defactinib, GSK2256098, PF-00562271, VS-4718 or a pharmaceutically acceptable salt thereof.

FIELD OF THE INVENTION

The present disclosure belongs to the field of pharmaceutical chemistry,and particularly relates to use of an FAK inhibitor in the manufactureof a medicament for treating tumors having an NRAS mutation.

BACKGROUND OF THE INVENTION

FAK, also known as protein tyrosine kinase 2 (PTK2), is a non-receptortyrosine kinase and a key component of the focal adhesion complex. FAKplays an important role in mediating integrin and growth factorsignaling to regulate invasion, proliferation and survival of tumorcells. FAK is widely expressed and evolutionarily conserved. Studies inthe past 20 years have shown that FAK is overexpressed in a variety ofsolid tumors, and the expression level is negatively correlated withtumor prognosis. Recent studies have also shown that FAK plays animportant role in regulating the tumor microenvironment, which suggeststhat FAK plays an important role in adaptive resistance to immunotherapyand anti-tumor therapy. Both in vitro and in vivo preclinical studieshave shown that blocking FAK has antitumor effects. However, theinhibitory activity of FAK inhibitors against tumors varies widely.

BI853520 is a FAK inhibitor. In human tumor CDX (Cell line-DerivedXenograft) mice model of 37 different tumors, BI853520 has showeddifferent anti-tumor activities, and its TGI (tumor growth inhibition)span from 0 to 107%. Inhibiting tumor growth by using BI853520 in a moretargeted manner is a technical problem to be solved urgently in thisfield.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides use of a FAK inhibitor inthe manufacture of a medicament for treating a tumor having an NRASmutation.

Optionally, the FAK inhibitor is BI853520, defactinib, GSK2256098,PF-00562271, VS-4718 or a pharmaceutically acceptable salt thereof.

Alternatively, the FAK inhibitor is BI853520 or a pharmaceuticallyacceptable salt thereof, especially BI853520 tartrate.

The BI 853520 has the following structure:

Optionally, the medicament is used in combination with an effectiveamount of a second therapeutic agent.

Optionally, the medicament is used in combination with radiotherapy orcell therapy.

Optionally, the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma,non-small cell lung cancer, small cell lung cancer, hepatocellularcarcinoma, cholangiocarcinoma, myelodysplastic syndrome, acutelymphoblastic leukemia, acute myeloid leukemia, chronic myeloidleukemia, thyroid cancer, glioma, colon cancer, rectal cancer,colorectal cancer, ovarian cancer, bladder cancer, prostate cancer,breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma,leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, headand neck cancer, stomach cancer, esophageal cancer, gastroesophagealjunction cancer, thymic cancer, pancreatic cancer, endometrial cancer,cervical cancer, melanoma, skin cancer, germ cell carcinoma,nasopharyngeal carcinoma, oropharyngeal carcinoma or laryngealcarcinoma; further the tumor is acute myeloid leukemia, melanoma,thyroid carcinoma, colorectal carcinoma, esophageal carcinoma,hepatocellular carcinoma, ovarian carcinoma, fibrosarcoma orcholangiocarcinoma.

Optionally, the second therapeutic agent is one or more selected fromchemotherapeutic agents, targeted therapeutic agents and immunotherapyagents.

Optionally, the second therapeutic agent is one or more selected fromthe group consisting of nimustine, carmustine, lomustine, temozolomide,cyclophosphamide, isocyclophosphamide, glyfosfin, doxifluridine,Furtulon, fluorouraeil, mercaptopurine, azathioprine, tioguanine,floxuridine, tegafur, gemcitabine, decitabine, carmofur, hydroxyurea,methotrexate, UFT, capecitabine, ancitabine, thiotepa, actinomycin D,adriamycin, liposomal doxorubicin, daunorubicin, epirubicin, mitomycin,pingyangmycin, pirarubicin, valrubicin, idarubicin, irinotecan,harringtonine, camptothecin, hydroxycamptothecine, topotecan,vinorelbine (navelbine), taxol, taxotere, hycamtin, vinblastine,vincristine, vindesine, vindesine sulfate, vincaleukoblastine,teniposide, etoposide, elemene, atamestane, anastrozole,aminoglutethimide, letrozole, formestane, megestrol, tamoxifen,asparaginase, carboplatin, cisplatin, dacarbazine, oxaliplatin,eloxatin, Eloxatin, mitoxantrone, procarbazine, docetaxel, gefitinib,erlotinib, icotinib, afatinib, osimertinib, crizotinib, ceritinib,alectinib, lapatinib, everolimus, palbociclib, ribociclib, apatinib,regorafenib, sorafenib, sunitinib, temsirolimus, lenvatinib, pazopanib,Alectinib, axitinib, cabozantinib, trametinib, binimetinib, vemurafenib,dabrafenib, Cobimetinib, vandetanib, bortezomib, palbociclib,lenalidomide, ixazomib, imatinib, dasatinib, bosutinib, ponatinib,ibrutinib, idelalisib, belinostat, romidepsin, vorinostat, olaparib,niraparib, denosumab, vismodegib, sonidegib, rucaparib, brigatinib,bicalutamide, enzalutamide, abiraterone, abemaciclib, apalutamide,aflibercept, azacitidine, bleomycin, chlorambucil, cytarabine,Asparaginase, epothilone, fludarabine, flutamide, mechlorethamine,paclitaxel, pemetrexed, raltitrexed, necitumumab, bevacizumab,ramucirumab, Ado-trastuzumab, pertuzumab, cetuximab, panitumumab,alirocumab, durvalumab, nimotuzumab, daratumumab, atezolizumab,sintilimab, toripalimab, camrelizumab, tislelizumab, durvalumab,nivolumab, and pembrolizumab.

Optionally, the second therapeutic agent is selected from the groupconsisting of decitabine, gemcitabine, cisplatin, carboplatin,oxaliplatin, adriamycin, liposomal doxorubicin, taxol, docetaxel,trametinib, binimetinib, cobimetinib, durvalumab, atezolizumab,sintilimab, toripalimab, camrelizumab, tislelizumab, nivolumab, andpembrolizumab.

Optionally, the second therapeutic agent is docetaxel, liposomaldoxorubicin, cobimetinib, pembrolizumab, or decitabine.

Optionally, the second therapeutic agent is cobimetinib.

In another aspect, the present disclosure also provides a method oftreating a tumor having an NRAS mutation comprising administering to anindividual an effective amount of a FAK inhibitor.

Optionally, the FAK inhibitor is BI853520 or defactinib, GSK2256098,PF-00562271, VS-4718 or a pharmaceutically acceptable salt thereof,alternatively, the FAK inhibitor is BI853520 or a pharmaceuticallyacceptable salt thereof, especially BI853520 tartrate.

Optionally, the method further comprises administering to the individualan effective amount of a second therapeutic agent.

Optionally, the method further includes radiotherapy or cell therapy.

Optionally, the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma,non-small cell lung cancer, small cell lung cancer, hepatocellularcarcinoma, cholangiocarcinoma, myelodysplastic syndrome, acutelymphoblastic leukemia, acute myeloid leukemia, chronic myeloidleukemia, thyroid cancer, glioma, colon cancer, rectal cancer,colorectal cancer, ovarian cancer, bladder cancer, prostate cancer,breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma,leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, headand neck cancer, stomach cancer, esophageal cancer, gastroesophagealjunction cancer, thymic cancer, pancreatic cancer, endometrial cancer,cervical cancer, melanoma, skin cancer, germ cell carcinoma,nasopharyngeal carcinoma, oropharyngeal carcinoma or laryngealcarcinoma; further the tumor is acute myeloid leukemia, melanoma,thyroid carcinoma, colorectal carcinoma, esophageal carcinoma,hepatocellular carcinoma, ovarian carcinoma, fibrosarcoma orcholangiocarcinoma.

Optionally, the second therapeutic agent is one or more selected fromchemotherapeutic agents, targeted therapeutic agents and immunotherapyagents.

Optionally, the second therapeutic agent is one or more selected fromthe group consisting of nimustine, carmustine, lomustine, temozolomide,cyclophosphamide, isocyclophosphamide, glyfosfin, doxifluridine,Furtulon, fluorouraeil, mercaptopurine, azathioprine, tioguanine,floxuridine, tegafur, gemcitabine, decitabine, carmofur, hydroxyurea,methotrexate, UFT, capecitabine, ancitabine, thiotepa, actinomycin D,adriamycin, liposomal doxorubicin, daunorubicin, epirubicin, mitomycin,pingyangmycin, pirarubicin, valrubicin, idarubicin, irinotecan,harringtonine, camptothecin, hydroxycamptothecine, topotecan,vinorelbine (navelbine), taxol, taxotere, hycamtin, vinblastine,vincristine, vindesine, vindesine sulfate, vincaleukoblastine,teniposide, etoposide, elemene, atamestane, anastrozole,aminoglutethimide, letrozole, formestane, megestrol, tamoxifen,asparaginase, carboplatin, cisplatin, dacarbazine, oxaliplatin,eloxatin, Eloxatin, mitoxantrone, procarbazine, docetaxel, gefitinib,erlotinib, icotinib, afatinib, osimertinib, crizotinib, ceritinib,alectinib, lapatinib, everolimus, palbociclib, ribociclib, apatinib,regorafenib, sorafenib, sunitinib, temsirolimus, lenvatinib, pazopanib,Alectinib, axitinib, cabozantinib, trametinib, binimetinib, vemurafenib,dabrafenib, Cobimetinib, vandetanib, bortezomib, palbociclib,lenalidomide, ixazomib, imatinib, dasatinib, bosutinib, ponatinib,ibrutinib, idelalisib, belinostat, romidepsin, vorinostat, olaparib,niraparib, denosumab, vismodegib, sonidegib, rucaparib, brigatinib,bicalutamide, enzalutamide, abiraterone, abemaciclib, apalutamide,aflibercept, azacitidine, bleomycin, chlorambucil, cytarabine,Asparaginase, epothilone, fludarabine, flutamide, mechlorethamine,paclitaxel, pemetrexed, raltitrexed, necitumumab, bevacizumab,ramucirumab, Ado-trastuzumab, pertuzumab, cetuximab, panitumumab,alirocumab, durvalumab, nimotuzumab, daratumumab, atezolizumab,sintilimab, toripalimab, camrelizumab, tislelizumab, durvalumab,nivolumab, and pembrolizumab.

Optionally, the second therapeutic agent is selected from the groupconsisting of decitabine, gemcitabine, cisplatin, carboplatin,oxaliplatin, adriamycin, liposomal doxorubicin, taxol, docetaxel,trametinib, binimetinib, cobimetinib, durvalumab, atezolizumab,sintilimab, toripalimab, camrelizumab, tislelizumab, nivolumab, andpembrolizumab.

Optionally, the second therapeutic agent is docetaxel, liposomaldoxorubicin, cobimetinib, pembrolizumab, or decitabine.

Optionally, the second therapeutic agent is cobimetinib.

Optionally, the second therapeutic agent is administered simultaneously,alternately or sequentially.

Optionally, the FAK inhibitor and radiotherapy or cell therapy areperformed simultaneously, alternately or sequentially.

In yet another aspect, the present disclosure provides a FAK inhibitorfor the treatment of a tumor having an NRAS mutation.

Optionally, the FAK inhibitor is BI853520, defactinib, GSK2256098,PF-00562271, VS-4718 or a pharmaceutically acceptable salt thereof,alternatively, the FAK inhibitor is BI853520 or a pharmaceuticallyacceptable salt thereof, especially BI853520 tartrate.

Optionally, the FAK inhibitor is further used in combination with aneffective amount of a second therapeutic agent.

Optionally, the FAK inhibitor is further used in combination withradiotherapy or cell therapy.

Optionally, the tumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma,non-small cell lung cancer, small cell lung cancer, hepatocellularcarcinoma, cholangiocarcinoma, myelodysplastic syndrome, acutelymphoblastic leukemia, acute myeloid leukemia, chronic myeloidleukemia, thyroid cancer, glioma, colon cancer, rectal cancer,colorectal cancer, ovarian cancer, bladder cancer, prostate cancer,breast cancer, liposarcoma, fibrosarcoma, rhabdomyosarcoma,leiomyosarcoma, angiosarcoma, neuroblastoma, renal cell carcinoma, headand neck cancer, stomach cancer, esophageal cancer, gastroesophagealjunction cancer, thymic cancer, pancreatic cancer, endometrial cancer,cervical cancer, melanoma, skin cancer, germ cell carcinoma,nasopharyngeal carcinoma, oropharyngeal carcinoma or laryngealcarcinoma; further the tumor is acute myeloid leukemia, melanoma,thyroid carcinoma, colorectal carcinoma, esophageal carcinoma,hepatocellular carcinoma, ovarian carcinoma, fibrosarcoma orcholangiocarcinoma.

Optionally, the second therapeutic agent is one or more selected fromchemotherapeutic agents, targeted therapeutic agents and immunotherapyagents.

Optionally, the second therapeutic agent is one or more selected fromthe group consisting of nimustine, carmustine, lomustine, temozolomide,cyclophosphamide, isocyclophosphamide, glyfosfin, doxifluridine,Furtulon, fluorouraeil, mercaptopurine, azathioprine, tioguanine,floxuridine, tegafur, gemcitabine, decitabine, carmofur, hydroxyurea,methotrexate, UFT, capecitabine, ancitabine, thiotepa, actinomycin D,adriamycin, liposomal doxorubicin, daunorubicin, epirubicin, mitomycin,pingyangmycin, pirarubicin, valrubicin, idarubicin, irinotecan,harringtonine, camptothecin, hydroxycamptothecine, topotecan,vinorelbine (navelbine), taxol, taxotere, hycamtin, vinblastine,vincristine, vindesine, vindesine sulfate, vincaleukoblastine,teniposide, etoposide, elemene, atamestane, anastrozole,aminoglutethimide, letrozole, formestane, megestrol, tamoxifen,asparaginase, carboplatin, cisplatin, dacarbazine, oxaliplatin,eloxatin, Eloxatin, mitoxantrone, procarbazine, docetaxel, gefitinib,erlotinib, icotinib, afatinib, osimertinib, crizotinib, ceritinib,alectinib, lapatinib, everolimus, palbociclib, ribociclib, apatinib,regorafenib, sorafenib, sunitinib, temsirolimus, lenvatinib, pazopanib,Alectinib, axitinib, cabozantinib, trametinib, binimetinib, vemurafenib,dabrafenib, Cobimetinib, vandetanib, bortezomib, palbociclib,lenalidomide, ixazomib, imatinib, dasatinib, bosutinib, ponatinib,ibrutinib, idelalisib, belinostat, romidepsin, vorinostat, olaparib,niraparib, denosumab, vismodegib, sonidegib, rucaparib, brigatinib,bicalutamide, enzalutamide, abiraterone, abemaciclib, apalutamide,aflibercept, azacitidine, bleomycin, chlorambucil, cytarabine,Asparaginase, epothilone, fludarabine, flutamide, mechlorethamine,paclitaxel, pemetrexed, raltitrexed, necitumumab, bevacizumab,ramucirumab, Ado-trastuzumab, pertuzumab, cetuximab, panitumumab,alirocumab, durvalumab, nimotuzumab, daratumumab, atezolizumab,sintilimab, toripalimab, camrelizumab, tislelizumab, durvalumab,nivolumab, and pembrolizumab.

Optionally, the second therapeutic agent is selected from the groupconsisting of decitabine, gemcitabine, cisplatin, carboplatin,oxaliplatin, adriamycin, liposomal doxorubicin, taxol, docetaxel,trametinib, binimetinib, cobimetinib, durvalumab, atezolizumab,sintilimab, toripalimab, camrelizumab, tislelizumab, nivolumab, andpembrolizumab.

Optionally, the second therapeutic agent is docetaxel, liposomaldoxorubicin, cobimetinib, pembrolizumab, or decitabine.

Optionally, the second therapeutic agent is cobimetinib.

Definition

The following terms and symbols used in this application have themeanings set forth below unless the context dictates otherwise.

The term “FAK inhibitor” as used herein refers to a potent inhibitor ofFAK, which may be suitable for mammals, particularly humans.

The term “NRAS” as used herein refers to an oncogene that is a member ofthe RAS oncogene family which also includes two other genes: KRAS andHRAS. These genes play important roles in cell division, celldifferentiation and apoptosis.

The term “NRAS mutation” as used herein means that when a pathogenicmutation occurs in the NRAS gene, the N-Ras protein encoded by it willbe in a state of continuous activation, resulting in uncontrolled cellproliferation and tumor formation.

The term “treating” as used herein refers to the administration of oneor more drugs, particularly a FAK inhibitor described herein, especiallyBI853520 or a pharmaceutically acceptable salt thereof, to an individualwith a disease or symptoms of said disease, to cure, alleviate, reduce,alter, remedy, ameliorate, improve or affect the disease or the symptomsof the disease. In some embodiments, the disease is a tumor or cancer.In further embodiments, the disease is a cancer or tumor having an NRASmutation.

The term “tumor” as used herein refers to an abnormal lesion formed byabnormally clonal proliferation of cells of local tissues which lose thenormal regulation on their growth at the gene level under the action ofvarious tumorigenic factors. Examples include, but are not limited to:Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lung cancer,small cell lung cancer, hepatocellular carcinoma, cholangiocarcinoma,myelodysplastic syndrome, acute lymphoblastic leukemia, acute myeloidleukemia, chronic myeloid leukemia, thyroid cancer, glioma, coloncancer, rectal cancer, colorectal cancer, ovarian cancer, bladdercancer, prostate cancer, breast cancer, liposarcoma, fibrosarcoma,rhabdomyosarcoma, leiomyosarcoma, angiosarcoma, neuroblastoma, renalcell carcinoma, head and neck cancer, stomach cancer, esophageal cancer,gastroesophageal junction cancer, thymic cancer, pancreatic cancer,endometrial cancer, cervical cancer, melanoma, skin cancer, germ cellcarcinoma, nasopharyngeal carcinoma, oropharyngeal carcinoma orlaryngeal carcinoma; further the tumors are acute myeloid leukemia,melanoma, thyroid carcinoma, colorectal carcinoma, esophageal carcinoma,hepatocellular carcinoma, ovarian carcinoma, fibrosarcoma andcholangiocarcinoma.

As used herein, the term “individual” refers to mammals and non-mammals.Mammals means any member of the mammalian class including, but notlimited to, humans; non-human primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, horses, sheep,goats, and swine; domestic animals such as rabbits, dogs, and cats;laboratory animals including rodents, such as rats, mice, and guineapigs; and the like. Examples of non-mammals include, but are not limitedto, birds, and the like. The term “individual” does not denote aparticular age or sex. In some embodiments, the individual is a human.

As used herein, the term “pharmaceutically acceptable” means non-toxic,biologically tolerable and suitable for administration to an individual.

As used herein, the term “pharmaceutically acceptable salt” refers to anacid addition salt of BI853520 that is non-toxic, biologically tolerableand suitable for administration to an individual. The “pharmaceuticallyacceptable salt” includes, but are not limited to, acid addition saltsformed by BI853520 with an inorganic acid, such as hydrochloride,hydrobromide, carbonate, bicarbonate, phosphate, sulfate, sulfite,nitrate, and the like; and acid addition salts formed by BI853520 withan organic acid, such as formate, acetate, malate, maleate, fumarate,tartrate, succinate, citrate, lactate, methanesulfonate,p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate,stearate, and salts with alkane-dicarboxylic acid of formulaHOOC—(CH₂)_(n)—COOH (wherein n is 0-4), etc. In some embodiments, thesalt is a tartrate salt.

In addition, a pharmaceutically acceptable acid addition salt may beproduced by dissolving the free base in a suitable solvent and treatingthe solution with an acid, in accordance with conventional proceduresfor preparing acid addition salts from basic compounds. Those skilled inthe art can determine, without undue experimentation, a variety ofsynthetic methods, which are used to prepare non-toxic pharmaceuticallyacceptable acid addition salts.

As used herein, the term “effective amount” refers to an amountsufficient to generally bring about a beneficial effect in anindividual. The effective amount of the compounds of the presentdisclosure may be ascertained by conventional methods (such as modeling,dose escalation studies or clinical trials), and by taking intoconsideration conventional influencing factors (such as the mode ofadministration, the pharmacokinetics of the compound, the severity andcourse of the disease, the individual's medical history, theindividual's health status and the individual's response to drugs).

The term “second therapeutic agent” as used herein refers to one or moredrugs used to prevent and/or treat diseases. In some embodiments, thesecond therapeutic agent is one or more selected from chemotherapeuticagents, targeted therapeutic agents and immunotherapeutic agents. Insome embodiments, the second therapeutic agent is one or more selectedfrom the group consisting of nimustine, carmustine, lomustine,temozolomide, cyclophosphamide, isocyclophosphamide, glyfosfin,doxifluridine, Furtulon, fluorouraeil, mercaptopurine, azathioprine,tioguanine, floxuridine, tegafur, gemcitabine, decitabine, carmofur,hydroxyurea, methotrexate, UFT, capecitabine, ancitabine, thiotepa,actinomycin D, adriamycin, liposomal doxorubicin, daunorubicin,epirubicin, mitomycin, pingyangmycin, pirarubicin, valrubicin,idarubicin, irinotecan, harringtonine, camptothecin,hydroxycamptothecine, topotecan, vinorelbine (navelbine), taxol,taxotere, hycamtin, vinblastine, vincristine, vindesine, vindesinesulfate, vincaleukoblastine, teniposide, etoposide, elemene, atamestane,anastrozole, aminoglutethimide, letrozole, formestane, megestrol,tamoxifen, asparaginase, carboplatin, cisplatin, dacarbazine,oxaliplatin, eloxatin, Eloxatin, mitoxantrone, procarbazine, docetaxel,gefitinib, erlotinib, icotinib, afatinib, osimertinib, crizotinib,ceritinib, alectinib, lapatinib, everolimus, palbociclib, ribociclib,apatinib, regorafenib, sorafenib, sunitinib, temsirolimus, lenvatinib,pazopanib, Alectinib, axitinib, cabozantinib, trametinib, binimetinib,vemurafenib, dabrafenib, Cobimetinib, vandetanib, bortezomib,palbociclib, lenalidomide, ixazomib, imatinib, dasatinib, bosutinib,ponatinib, ibrutinib, idelalisib, belinostat, romidepsin, vorinostat,olaparib, niraparib, denosumab, vismodegib, sonidegib, rucaparib,brigatinib, bicalutamide, enzalutamide, abiraterone, abemaciclib,apalutamide, aflibercept, azacitidine, bleomycin, chlorambucil,cytarabine, Asparaginase, epothilone, fludarabine, flutamide,mechlorethamine, paclitaxel, pemetrexed, raltitrexed, necitumumab,bevacizumab, ramucirumab, Ado-trastuzumab, pertuzumab, cetuximab,panitumumab, alirocumab, durvalumab, nimotuzumab, daratumumab,atezolizumab, sintilimab, toripalimab, camrelizumab, tislelizumab,durvalumab, nivolumab, and pembrolizumab. Optionally, the secondtherapeutic agent is selected from decitabine, gemcitabine, cisplatin,carboplatin, oxaliplatin, adriamycin, liposomal doxorubicin, taxol,docetaxel, trametinib, binimetinib, cobimetinib, durvalumab,atezolizumab, sintilimab, toripalimab, camrelizumab, tislelizumab,nivolumab, and pembrolizumab. Optionally, the second therapeutic agentis docetaxel, liposomal doxorubicin, cobimetinib, pembrolizumab, ordecitabine. Optionally, the second therapeutic agent is cobimetinib.

In addition, the drug substances from which the second therapeutic agentis selected may be slightly different in names due to differenttranslations, but they still refer to one drug substance, for example:cobimetinib may be translated into Chinese as Ka Bi Ti Ni or Kao Bi TiNi.

The term “inhibition” as used herein refers to a reduction in thebaseline activity of a biological activity or process.

Technical and scientific terms used herein without specific definitionhave the meaning commonly understood by those skilled in the art towhich the invention pertains.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows influence of IL-3 on the antiproliferative effect of BI853520 in the Ba/F3-NRAS^(G12D) cell line. It illustrates that forBI853520, the IC₅₀ was increased from 3.4 to 15 μM after addition ofIL-3 in NRAS^(G12D)-transfected Ba/F3 cell line (increased by more than4-fold). GraphPad Prism 6 software and Student's t-test were used forstatistical data analysis. Statistically significant differencethreshold was set at P=0.05.

FIG. 2 shows changes in body weight. It illustrates that animals in thecontrol group lost 1.9% body weight; animals treated with BI 853520 at 6mg/kg had a median weight gain of 4.9% (p=0.9966 compared to the controlgroup); animals treated with 853520 at 12.5 mg/kg had a median weightgain of 5.7% (p=0.9996 compared to the control group); animals treatedwith BI 853520 at 25 mg/kg had a median weight gain of 9.4% (p=1.0000compared to the control group); and animals treated with BI 853520 at 50mg/kg had a median weight gain of 8.3% (p=0.999 compared to the controlgroup).

FIG. 3 shows HT-1080 tumor growth kinetics. It illustrates that on day13, the median tumor volume of the control group was 823 mm³; the mediantumor volume of the group treated with BI 853520 at 6 mg/kg was 322 mm³;the median tumor volume of the group treated with BI 853520 at 12.5mg/kg was 149 mm³; the median tumor volume of the group treated with BI853520 at 25 mg/kg was 15 mm³; and the median tumor volume of the grouptreated with BI 853520 at 50 mg/kg was 322 mm³.

FIG. 4 shows KYSE-270 tumor growth kinetics, which illustrates themedian tumor volume increased from 179 mm³ to 1032 mm³ in the controlgroup. Compared to the control group, the median tumor volume of thegroup treated with 50 mg/kg BI 853520 per day decreased from 175 mm³ to126 mm³. Treatment with 10 mg/kg paclitaxel once a week had no effect ontumor growth, in which the median tumor volume increased from 190 mm³ to1033 mm³. The combination therapy of 50 mg/kg BI 853520 once a day with10 mg/kg paclitaxel once a week significantly delayed tumor growth, withthe median tumor volume decreased from 173 mm³ to 87 mm³.

FIG. 5 shows changes in body weight. It illustrates that animals in thecontrol group had a 10.3% reduction in the median body weight, andcompared to the control group, animals treated with 50 mg/kg BI 853520daily had a 2.8% increase in the median body weight, animals treatedwith 10 mg/kg paclitaxel once a week had a 9.7% reduction in the medianbody weight, and animals treated with the combination therapy of 50mg/kg BI 853520 once daily with 10 mg/kg paclitaxel once a week had a3.5% increase in the median body weight.

FIG. 6 shows GAK tumor growth kinetics. It illustrates that in the GAKmodel, daily treatment with BI 853520 delayed tumor growth compared tothe control group.

FIG. 7 shows HMVII tumor growth kinetics. It illustrates that in theHMVII model, daily treatment with BI 853520 delayed tumor growthcompared to the control group.

DETAILED DESCRIPTION OF THE INVENTION

The following examples are provided to further illustrate the presentdisclosure. It should be understood that these examples are only used toillustrate the present disclosure and not to limit the scope of thepresent disclosure.

The experimental methods without specific conditions in the followingexamples can be carried out according to the conventional conditions ofthis type of reaction or according to the conditions suggested by themanufacturers.

The experimental materials and reagents used in the following examplescan be obtained from commercial sources unless otherwise specified.

The meanings of the abbreviations used in the examples are as follows:

ATCC American Type Culture Collection

CO₂ Carbon dioxide

d Days

FCS Fetal Calf Serum

FBS Fetal Bovine Serum

g Gram

kg Kilograms

l Liter

mg Milligram

ml Milliliter

μl Microliter

mM Millimole

mm³ Cubic millimeter

GLP Good Laboratory Practice

h Hours

MCB Master Cell Bank

mean Arithmetic average

MTD Maximum tolerated dose

n Sample number

PBS Phosphate Buffered Saline

RPMI 1640 RPMI 1640 Medium

p.o. peros

qd Daily

q7d Every 7 days (once a week)

s.c. Subcutaneous

SD Standard deviation

TGI Tumor Growth Inhibition

WCB Working Cell Bank

V_(rel) Relative Tumor Volume (relative to Day 1)

Example 1: Correlation Between Antitumor Effect of B1853520 and NRAS

1. Materials and Method:

1.1. DNA Construction and Retrovirus Production

Retroviral constructs expressing NRAS-G12D, KRAS4A-G12D, KRAS4B-G12D,and NRAS-G12D-C1815 as N-terminal GFP fusion proteins were preparedaccording to a method as described in Cuiffo B, Ren R. Palmitoylation ofoncogenic NRAS is essential for leukemogenesis. Blood., 2010, 115:3598-605, and Parikh C, Subrahmanyam R, Ren R. Oncogenic NRAS, KRAS, andHRAS exhibit different leukemogenic potentials in mice. Cancer Res.,2007, 67: 7139-46. Retroviruses were prepared by using Bosc23 cells andthe titre determination was performed by the method described in ParikhC, Subrahmanyam R, Ren R. Oncogenic NRAS, KRAS, and HRAS exhibitdifferent leukemogenic potentials in mice. Cancer Res., 2007, 67:7139-46.

1.2. Cell Culture and Retroviral Transduction

The Ba/F3 cell line was obtained from ATCC in 2009, and culturedaccording to a method described in Zhang X, Ren R. Bcr-abl efficientlyinduces a myeloproliferative disease and production of excessinterleukin-3 and granulocyte-macrophage colony-stimulating factor inmice: A novel model for chronic myelogenous leukemia. Blood., 1998, 92:3829-40. Growth factor-dependent BA/F3 cells were cultured in RPMI 1640including 10% FBS and supplemented with 15% WEHI-3 conditioned medium orrecombinant IL-3 (Roche) as a source of IL-3 at a final concentration of1 ng/ml. BA/F3 cells were transduced with retrovirus according to amethod as described in Parikh C, Subrahmanyam R, Ren R. Oncogenic NRAS,KRAS, and HRAS exhibit different leukemogenic potentials in mice. CancerRes., 2007, 67: 7139-46 and Fredericks J, Ren R. The role of RASeffectors in BCR/ABL induced chronic myelogenous leukemia. Front MedChina., 2013, 7: 452-61. Upon receipt of the original stock, cells wereexpanded and frozen at a low passage rate (<3). Frozen cells were thawedand used in this study, with cell passages limited to 15 for allexperimental procedures. All cells were tested regularly to ensurefreedom and maintenance in a normal state of mycoplasma.

1.3. Materials

BI853520: synthesized according to the method in the patentWO2010058032.

1.4. Antiproliferative Test

NRAS^(G12D)-transfected Ba/F3 (Ba/F3-N) or KRAS^(G12D)-transfected Ba/F3(Ba/F3-N) cells were seeded into 96-well cell culture cluster plate in100 μL of medium at a density of 5,000 cells per well. BI853520 wasdiluted 3-fold with RPMI 1640 at 9 points, and added according to thevolume of each well, starting at 10 μmol/L to reach a 3-foldconcentration gradient (final solvent concentration <1/1000). PD0325901was used as a positive control. After 48 hours of incubation, cellviability was measured using CellTiter Glo (Promega). Luminescence wasdetected using an Envision plate reader (PerkinElmer). The calculationmethod of Z factor was as described in Zhang J H, Chung T D Y, OldenburgK R. A simple statistical parameter for use in evaluation and validationof high throughput screening assays. J Biomol Screen., 1999, 4: 67-73,and the following analysis only used data with Z factor >0.6.Dose-response curves were fitted based on the relative percentage ofviable cells in nonlinear fitness (curve fitting) using GraphPad Prism 6software (http://www.graphpad.com/scientific-software/prism/). Theinternal software analysis “non-linear regression (curve fitting)” andthe equation “log (inhibitor) vs. slope of response variable” were usedfor data analysis and IC₅₀ calculation.

2. Statistical Analysis:

GraphPad Prism 6 software and Student's t-test were used for statisticaldata analysis. Statistically significant difference threshold was set atP=0.05.

3. Results:

Ba/F3 is a murine marrow-derived cell line depending on IL-3 forsurvival and proliferation, and has been successfully used inhigh-throughput assays for kinase drug discovery. We tested this systemwith oncogenic RAS and found that oncogenic NRAS (NRAS^(G12D)) couldconvert Ba/F3 cells to being independent on IL-3. FIG. 1 and Table 1show the antiproliferative effect of BI853520 on Ba/F3-NRAS^(G12D) cellline with IC₅₀ value of 3.4 μM. The IC₅₀ increased more than 4-foldafter the addition of IL-3, indicating that the inhibitory effect wasrelated to NRAS.

TABLE 1 Influence of adding IL-3 or not on the antiproliferative effectof BI853520 in Ba/F3-NRAS^(G12D) cell line IC₅₀ (μM) Cell line Compound−IL-3 +IL-3 Ba/F3-NRAS^(G12D) BI853520 3.4 15

Example 2: The Effect of B1853520 in a Mouse Model of Human Fibrosarcoma(Cell Line HT-1080)

1. Materials and Method:

1.1. Design of Research

Treatment began when the median tumor volume was between 70-130 mm³.

10 mice in the control group, and 7 mice in the treatment group.

Oral gavage once daily.

Control: 0.5% hydroxyethyl cellulose

BI 853520: 50 mg/kg, 25 mg/kg, 12.5 mg/kg, 6 mg/kg,

Tumor volume was measured 3 times a week and body weight was monitoreddaily.

Evaluation of treatment outcome was based on absolute volume ofindividual tumors.

Tolerability was assessed based on changes in body weight.

1.2. Materials

BI853520: synthesized according to the method in the patentWO2010058032. The dry powder was suspended in 0.5% hydroxyethylcellulose to obtain the desired concentration for each assay. The pH ofthis formulation was 3.5.

HT-1080 cells: HT-1080 cells carrying NRAS, CDKN2A and IDH1 genemutations were obtained from ATCC (CRL-121). Cells were grown in T175tissue culture flasks with DMEM+Glutamax supplemented with 10%heat-inactivated fetal bovine serum as medium. Cells were cultured at37° C. and 5% CO₂ in humidified air.

Mice: Athymic female BomTac, approximately 6-week-old NMRI-Foxn1^(nu)mice were purchased from Taconic, Denmark. After arriving in the animalroom, mice were acclimated to the new environment for at least 3 daysbefore being used for assay. The animals were housed under standardconditions (temperature 21.5±1.5° C. and 55±10% humidity) with 5 mice ineach group. A standard diet and autoclaved tap water were provided forad libitum feeding. A Datamars T-IS 8010 FDX-B transponder implantedsubcutaneously in the neck region and a LabMax II fixed reader were usedto identify each mouse. The cage card showed study number, animalidentification number, compound and dose level, route of administration,and dosing schedule for animals throughout the assay.

1.3. Random Establishment of Tumors:

To establish subcutaneous tumors, HT-1080 cells were harvested bytrypsinization, centrifuged, washed and suspended in ice-cold PBS+1×10⁸cells/ml. Then 100 μl of cell suspension containing 1×10⁷ cells wasinjected subcutaneously into the right flank of nude mice (1 site permouse). When tumors were established and reached a diameter of 5-8 mm (7days after cell injection), mice were randomly assigned to the treatmentgroup and the control group.

1.4. Dosing:

BI853520 was suspended in 0.5% hydroxyethyl cellulose, which wasadministered intragastrically through gavage needle every day, and thedosage was 10 mL/Kg.

1.5. Monitoring Tumor Growth and Side Effects:

Tumor diameters were measured with calipers three times a week (Monday,Wednesday, and Friday). The volume of each tumor [in mm³] was calculatedaccording to the equation, “tumor volume=length×diameter²π/6”. Tomonitor the side effects of the treatment, the mice were checked dailyfor abnormalities and their body weights were measured daily. Animalswere sacrificed at the end of the study (approximately two weeks afterthe start of treatment). During the study, animals with tumor necrosisor tumors larger than 2000 mm³ were sacrificed ahead of schedule forethical reasons.

2. Statistical Analysis:

At the end of the assay on day 13, statistical evaluation of tumorvolume and body weight parameters was performed. Absolute tumor volumeand percent change in body weight (referenced to initial weight onday 1) were used. A nonparametric approach was used, and the number ofobservations, median, minimum and maximum values were calculated. For aquick overview of possible treatment effects, the median tumor volumefor each treatment group T and the median tumor volume for the controlgroup C were used to calculate the TGI from day 1 to day d:

TGI=100×[(C _(d) −C ₁)−(T _(d) −T ₁)]/(C _(d) −C ₁)

wherein, C₁, T₁=median tumor volume in control and treatment groups atthe start of the assay (day 1).C_(d), T_(d)=median tumor volume in control and treatment groups at theend of the assay (day 13).

Each dose of test compound was compared to the control group using aone-sided descending wilcoxon test, taking reduction in tumor volume asa treatment effect and weight loss as a side effect. The P-values fortumor volume (the efficacy parameter) were compared and adjusted formultiple times according to Bonferroni-Holm, while the P-values for bodyweight (the tolerance parameter) were not adjusted so as not to overlookpossible side effects. Significance level was fixed at α=5%. A p-value(adjusted) of less than 0.05 was considered to show a statisticallysignificant difference between groups, and 0.05≤p-value<0.10 wasconsidered as an indicative difference. Statistical evaluations wereperformed using the software packages SAS version 9.2 (SAS InstituteInc., Cary, N.C., USA) and Proc StatXact version 8.0 (Cytel Software,Cambridge, Mass., USA).

3. Results:

3.1. During the test period, animals in the control group lost 1.9% bodyweight (FIG. 2 , Table 2), and on day 13, their median tumor volumereached 823 mm³ (FIG. 3 , Table 3).

3.2. Treatment with BI 853520 at 50 mg/kg once daily resulted in a TGIof 107% (p=0.0002), and tumor shrinkage was observed in all animals(FIG. 3 , Table 3). The median body weight of the animals increased by8.3% (p=0.999 compared to the control group) (FIG. 2 , Table 2).

3.3. Treatment with BI 853520 at 25 mg/kg once daily resulted in a TGIof 113% (p=0.0002) and tumor shrinkage in 6 animals (FIG. 3 , Table 3).The median body weight of the animals increased by 9.4% (p=1.0000compared to the control group) (FIG. 2 , Table 2).

3.4. Treatment with BI 853520 at 12.5 mg/kg once daily resulted in a TGIof 94% (p=0.0002) and tumor shrinkage in 2 animals (FIG. 3 , Table 3).The median body weight of the animals increased by 5.7% (p=0.9996compared to the control group) (FIG. 2 , Table 2).

3.5. Treatment with BI 853520 at 6 mg/kg once daily resulted in a TGI of70% (p=0.0004) and tumor shrinkage in 1 animal (FIG. 5 , Table 3). Themedian body weight of the animals increased by 4.9% (p=0.9966 comparedto the control group) (FIG. 2 , Table 2).

TABLE 2 Median weight change at the end of the assay Median DosageDosing weight change (mg/Kg) schedule (%) control group qd −1.9 50 qd+8.3 25 qd +9.4 12.5 qd +5.7 6 qd +4.7

TABLE 3 Median tumor volume Median tumor Tumor Dosage Dosing volumeshrinkage TGI (mg/Kg) schedule (mm³) [X/7] [%] control group qd 823 —n.a 50 qd 54 7 107 25 qd 15 6 113 12.5 qd 149 2 94 6 qd 322 1 70

4. Conclusion:

In the human HT-1080 fibrosarcoma model, BI 853520 demonstratedstatistically significant antitumor activity at all dose levels, andregression was observed in all the treatment groups; reduction in tumorvolume was observed in all animals at the highest dose level. Inprevious studies, the highest daily dose was 100 mg/kg without limitingtoxicity. Thus, significant efficacy was obtained at a dose at least 16times lower than the MTD (6 mg/kg).

Example 3: Antitumor Activity of BI 853520 in a Subcutaneous XenograftMouse Model Derived from the Human Esophageal Cancer Cell Line KYSE-270in NMRI Nude Mice

1. Materials and Methods

1.1. Model: Subcutaneous xenograft of human esophageal cancer cell lineKYSE-270 in NMRI nude mice.

1.2. Test compound: BI853520: synthesized according to the method inpatent WO2010058032.

1.3. Cells: KYSE-270 is an esophageal cancer cell line (Public HealthEngland, catalog number: 94072021). Cells were cultured in T175 tissueculture flasks containing 5% CO₂ at 37° C. The medium was RPMI 1640+HAMF12 (1:1) supplemented with 2% FCS and 2 mM Glutamax. Passage wasperformed three times a week at a ratio of 1:2.

1.4. Mice: Mice were 8 to 10 week old female mice (BomTac:NMRI-Foxn1^(nu)) purchased from Taconic, Denmark. Upon arrival in theanimal room, the mice were allowed to acclimate for at least 5 daysbefore being used for assay. The animals were housed under standardconditions (temperature: 21.5±1.5° C., humidity: 55+10%), 7 to 10 micefor each group. A standard diet and autoclaved tap water were providedfor ad libitum feeding. A subcutaneous microchip implanted underisoflurane anesthesia was used to identify each mouse. The cage cardshowed study number, animal identification number, compound and doselevel, route of administration, and dosing schedule for animalsthroughout the assay.

1.5. Random establishment of tumors: To establish subcutaneous tumors,KYSE-270 cells were harvested by centrifugation, washed and resuspendedin PBS+5% FCS at a concentration of 5×10⁷ cells/ml. Then 100 μl of cellsuspension containing 5×10⁶ cells was injected subcutaneously into theright flank of mice (1 site per mouse). When tumors were wellestablished and reached a volume of 94 to 252 mm³, mice were randomlyassigned to the treatment group and the control group (13 days aftercell injection).

1.6. Administration: BI 853520 was suspended in 0.5% hydroxyethylcellulose, which was administered intragastrically through gavage needleevery day, and the dosage was 10 mL/Kg body weight. Paclitaxel wasdissolved in saline (0.9% NaCl), which was administered intravenously ina volume of 10 ml/kg body weight. The suspension of BI853520 could beused for up to 7 days. Paclitaxel solution was stored at 6° C. and usedfor up to 14 days.

1.7. Monitoring tumor growth and side effects: Tumor diameters weremeasured with calipers three times a week. The volume [mm³] of eachtumor was calculated according to the equation “tumor volume=length xdiameter²×π/6”. To monitor the side effects of the treatment, the micewere checked daily for abnormalities and their body weights weremeasured three times a week. For ethical reasons, animals with tumorslarger than 1.5 cm in diameter, ulcerated tumors, or 20% body weightloss were euthanized.

2. Statistical Analysis:

Statistical assessment of tumor volume and body weight on day 13 wasperformed. Animal number 1 (control group) was excluded from thestatistical evaluation because of weight loss and had to be euthanizedearly (day 9). Measured tumor volume was used as the target variable inthe statistical analysis. The number of animals and the median, minimumand maximum tumor volumes in each group were calculated. For a quickoverview of possible treatment effects, the median tumor volume for eachtreatment group T and the median tumor volume for the control group Cwere used to calculate TGI:

TGI=100×[(C _(d) −C ₁)−(T _(d) −T ₁)]/(C _(d) −C ₁)

wherein, C₁, T₁=median tumor volume in control and treatment groups atthe start of the assay (day 1).

C_(d), T_(d)=median tumor volume in control and treatment groups at theend of the assay (day 13).

The wilcoxon test was used for comparison. For body weight, the percentchange relative to initial body weight on day 1 was used as the targetvariable for statistical analysis. The number of animals, and themedian, minimum and maximum weight changes in each group werecalculated.

One-sided test to compare all treatment groups with control group wasperformed for observing:

reduction in tumor volume (inhibition of tumor growth, efficacyparameter) andreduction in weight changes (weight loss, tolerance parameters).

Within each subtopic, p-values for the efficacy parameter were comparedand adjusted for multiple times according to Bonferroni-Holm. Thep-value for the tolerance parameter was kept constant so as not tooverlook possible side effects. Significance level was fixed at α=5%. Ap-value (adjusted) of less than 0.05 was considered to show astatistically significant difference between groups, and0.05≤p-value<0.10 was considered as an indicative difference.Statistical evaluations were performed using the SAS software packageversion 9.4 (SAS Institute Inc., Cary N.C., USA).

3. Results

Tumor Volume and Body Weight:

By day 13, the median tumor volume in the control group increased from179 mm³ to 1032 mm³ (FIG. 4 ). The median body weight of control animalsdecreased by 10.3% (FIG. 5 , Table 4). On day 9, one animal had to beeuthanized due to severe weight loss.

Daily treatment with 50 mg/kg BI 853520 significantly delayed tumorgrowth (median TGI=106%, p=0.0003) compared to the control group (FIG. 5, Table 4). On day 13, 6 of 7 tumors had shrunk (Table 4). The medianbody weight of the animals increased by 2.8%, which was notsignificantly different from the control group (p=0.9999) (FIG. 5 ,Table 4).

Treatment with 10 mg/kg paclitaxel once a week had no effect on tumorgrowth (median TGI=2%, p=0.3788) compared to the control group (FIG. 4 ,Table 4). On day 13, none of the animals had tumor shrinkage (Table 4).The median body weight of the animals decreased by 9.7%, which was notsignificantly different from the control group (p=0.7320) (FIG. 5 ,Table 4).

Combination therapy of BI 853520 at 50 mg/kg once daily and 10 mg/kgpaclitaxel once a week significantly delayed tumor growth (medianTGI=110%, p=0.0003) compared to the control group (FIG. 4 , Table 4). Onday 13, all animals had tumor shrinkage (Table 4). The median bodyweight of the animals increased by 3.5%, which was not significantlydifferent from the control group (p=1.0000) (FIG. 5 , Table 4).

TABLE 4 TGI, tumor shrinkage and body weight change in each group (day13) Dosage Tumor Change in (Dosing TGI shrinkage median weight CompoundSchedule) (%) [x/y] (%) Control group −(qd + q7d) −10.3 BI853520 50mg/Kg (qd)  106 6/7 +2.8 Paclitaxel 10 mg/Kg (q7d) 2 0/7 −9.7 BI853520 +50 mg/Kg (qd) + 110 7/7 3.5 Paclitaxel 10 mg/Kg (q7d)

4. Conclusion

In a subcutaneous xenografted model of the human esophageal cancer cellline KYSE-270, statistically significant inhibition of tumor growth wasobserved in the treatment group with 50 mg/kg BI 853520 and thetreatment group with 50 mg/kg BI 853520 in combination with 10 mg/kgpaclitaxel, and both were well tolerated.

Example 4: Tumor Growth Inhibitory Effect of BI 853520 on HMVII and GAKCDX Models (Melanoma Models with an NRAS Mutation)

1. Materials and Methods

1.1. Test compound: BI853520, which was synthesized according to themethod in the patent WO2010058032. BI853520 was formulated with 0.5%hydroxyethyl cellulose (Ashland).

1.2. Cells: The melanoma cell line GAK was purchased from JCRB CellBank, and HMVII cells were purchased from Sigma. GAK originated in theinguinal lymph nodes of patients with vaginal melanoma, whereas HMVIIoriginated in primary vaginal melanoma. GAK and HMVII cells werecultured in tissue culture flasks containing 5% CO₂ at 37° C. The mediawere Ham's F12 containing 10% heat-inactivated FBS and Ham's F10containing 15% FBS, respectively. All media were supplemented with 100U/mL penicillin, 100 mg/mL streptomycin, and 2 mM GlutaMAX. All cellculture reagents were purchased from GIBCO.

Both HMVII and GAK cell lines carried an NRAS mutation at the Q61position. HMVII (NRAS^(Q61K)) and GAK (NRAS^(Q61L)).

1.3. Mice: 4-week-old female balb/c nude mice (purchased from BeijingVital River Laboratory Animal Technology Co., Ltd.). After arriving inthe animal room, the mice were allowed to acclimate to the newenvironment for at least 2 weeks before being used for assay. Theanimals were housed under standard conditions (temperature: 21.5±1.5°C., humidity: 55±10%). A Standard diet and autoclaved tap water wereprovided ad libitum. All animal care and experimental procedures wereperformed in accordance with the animal care ethics guidelines approvedby the Medical Ethics Committee of Beijing Cancer Hospital and ResearchInstitute.

1.4. Random establishment of tumors:

To establish subcutaneous tumors, HMVII and GAK cells were harvested bytrypsinization, centrifuged, washed and resuspended in ice-cold PBS+5%FCS. Then 100 μl of the cell suspension containing 5×10⁶ cells wasinjected subcutaneously into the right flank of nude mice (1 site permouse). When tumors were well established and reached a median volume ofapproximately 400-600 mm³, mice were randomly assigned to the treatmentgroup and the control group.

1.5. Administration: BI 853520 was formulated in 0.5% hydroxyethylcellulose, which was intragastrically administered through gavage needleevery day, and the dosage was 10 mL/Kg body weight.

1.6. Monitoring tumor growth:

Tumor diameters were measured twice a week with calipers. The volume[mm³] of each tumor was calculated according to the equation “tumorvolume=length x diameter²×π/6”. Animals were sacrificed at the end ofthe study (approximately two weeks after the start of treatment). Duringthe study, animals with necrotic tumors or tumors exceeding 2000 mm³ insize were sacrificed for ethical reasons.

2. Statistical Analysis:

Tumor volumes were statistically assessed at the end of the assay. Fortumor volume, relative values were used.

The number of observations, and the median, minimum and maximum tumorvolumes were calculated. For a quick overview of possible treatmenteffects, the following metrics were calculated:

Relative Tumor Volume: (T/C)

${{T/C} = {100*\frac{T_{d}}{C_{d}}}};$

TGI from Day 1 to Day d:

TGI=100×[(C _(d) −C ₁)−(T _(d) −T ₁)]/(C _(d) −C ₁)

wherein, C₁, T₁=mean relative tumor volume in control and treatmentgroups at the start of the assay (day 1);

C_(d), T_(d)=mean relative tumor volume in control and treatment groupsat the end of the assay (day d).

Statistical evaluation was performed using the Student's t-test functionin Microsoft Excel, using two-tailed distribution and two-sample equalvariance type.

The significance level was fixed at α=5%. A p-value (adjusted) of lessthan 0.05 was considered to show a significant difference betweentreatment groups, and 0.05≤p-value<0.10 was considered as an indicativedifference.

3. Results

In the GAK model, daily treatment with BI 853520 delayed tumor growth(median TGI=80%, p=0.03) compared to the control group (FIG. 6 , Table5).

In the HMVII model, daily treatment with BI 853520 delayed tumor growth(median TGI=60%, p=0.17) compared to the control group (FIG. 7 , Table5).

TABLE 5 Mean tumor volume, mean TGI and P-value based on relative tumorvolume on day 14 Mean tumor Cell volume Mean Group line Scheme [mm³] TGIP A HMVII 0.5% hydroxyethyl 1237 ± 733  — — cellulose (n = 3) B HMVIIBI853520 671 ± 606 60% 0.17 50 mg/kg (n = 4) (during the 1^(st) week) 25mg/kg (during the 2^(nd) week) C GAK 0.5% hydroxyethyl 1387 ± 1180 — —cellulose (n = 3) D GAK BI853520 548 ± 144 80% 0.03 50 mg/kg (n = 3)(during the 1^(st) week) 25 mg/kg (during the 2^(nd) week)

4. Conclusion

BI853520 can inhibit tumor growth in CDX models of HMVII and GAK (twomelanoma models with an NRAS mutation).

All references mentioned herein are incorporated by reference in theirentirety, as if each were individually listed. It should be understoodthat after reading the disclosure of the present invention, thoseskilled in the art can make various changes or modifications to thepresent invention, and these equivalent forms also fall within the scopedefined by the appended claims of the present application.

1-7. (canceled)
 8. A method of treating tumors having an NRAS mutationcomprising administering to an individual an effective amount of a FAKinhibitor.
 9. The method of claim 8, wherein the FAK inhibitor isBI853520, defactinib, GSK2256098, PF-00562271, VS-4718 or apharmaceutically acceptable salt thereof, alternatively, the FAKinhibitor is BI853520 or a pharmaceutically acceptable salt thereof,especially BI853520 tartrate.
 10. The method of claim 8, furthercomprising administering to the individual an effective amount of asecond therapeutic agent.
 11. The method of claim 8, further comprisingradiotherapy or cell therapy.
 12. The method of claim 8, wherein thetumor is Hodgkin's lymphoma, non-Hodgkin's lymphoma, non-small cell lungcancer, small cell lung cancer, hepatocellular carcinoma,cholangiocarcinoma, myelodysplastic syndrome, acute lymphoblasticleukemia, acute myeloid leukemia, chronic myeloid leukemia, thyroidcancer, glioma, colon cancer, rectal cancer, colorectal cancer, ovariancancer, bladder cancer, prostate cancer, breast cancer, liposarcoma,fibrosarcoma, rhabdomyosarcoma, leiomyosarcoma, angiosarcoma,neuroblastoma, renal cell carcinoma, head and neck cancer, stomachcancer, esophageal cancer, gastroesophageal junction cancer, thymiccancer, pancreatic cancer, endometrial cancer, cervical cancer,melanoma, skin cancer, germ cell carcinoma, nasopharyngeal carcinoma,oropharyngeal carcinoma or laryngeal carcinoma.
 13. The method of claim10, wherein the second therapeutic agent is one or more selected fromchemotherapeutic agents, targeted therapeutic agents andimmunotherapeutic agents.
 14. The method of claim 10, wherein the secondtherapeutic agent is one or more selected from the group consisting ofnimustine, carmustine, lomustine, temozolomide, cyclophosphamide,isocyclophosphamide, glyfosfin, doxifluridine, Furtulon, fluorouraeil,mercaptopurine, azathioprine, tioguanine, floxuridine, tegafur,gemcitabine, decitabine, carmofur, hydroxyurea, methotrexate, UFT,capecitabine, ancitabine, thiotepa, actinomycin D, adriamycin, liposomaldoxorubicin, daunorubicin, epirubicin, mitomycin, pingyangmycin,pirarubicin, valrubicin, idarubicin, irinotecan, harringtonine,camptothecin, hydroxycamptothecine, topotecan, vinorelbine (navelbine),taxol, taxotere, hycamtin, vinblastine, vincristine, vindesine,vindesine sulfate, vincaleukoblastine, teniposide, etoposide, elemene,atamestane, anastrozole, aminoglutethimide, letrozole, formestane,megestrol, tamoxifen, asparaginase, carboplatin, cisplatin, dacarbazine,oxaliplatin, eloxatin, mitoxantrone, procarbazine, docetaxel, gefitinib,erlotinib, icotinib, afatinib, osimertinib, crizotinib, ceritinib,alectinib, lapatinib, everolimus, palbociclib, ribociclib, apatinib,regorafenib, sorafenib, sunitinib, temsirolimus, lenvatinib, pazopanib,axitinib, cabozantinib, trametinib, binimetinib, vemurafenib,dabrafenib, Cobimetinib, vandetanib, bortezomib, palbociclib,lenalidomide, ixazomib, imatinib, dasatinib, bosutinib, ponatinib,ibrutinib, idelalisib, belinostat, romidepsin, vorinostat, olaparib,niraparib, denosumab, vismodegib, sonidegib, rucaparib, brigatinib,bicalutamide, enzalutamide, abiraterone, abemaciclib, apalutamide,aflibercept, azacitidine, bleomycin, chlorambucil, cytarabine,epothilone, fludarabine, flutamide, mechlorethamine, paclitaxel,pemetrexed, raltitrexed, necitumumab, bevacizumab, ramucirumab,Ado-trastuzumab, pertuzumab, cetuximab, panitumumab, alirocumab,durvalumab, nimotuzumab, daratumumab, atezolizumab, sintilimab,toripalimab, camrelizumab, tislelizumab, durvalumab, nivolumab, andpembrolizumab.
 15. The method of claim 10, wherein the FAK inhibitor andthe second therapeutic agent are administered simultaneously,alternately or sequentially.
 16. The method of claim 11, wherein the FAKinhibitor and radiotherapy or cell therapy are performed simultaneously,alternately or sequentially. 17-23. (canceled)
 24. The method of claim12, wherein the tumor is acute myeloid leukemia, melanoma, thyroidcarcinoma, colorectal carcinoma, esophageal carcinoma, hepatocellularcarcinoma, ovarian carcinoma, fibrosarcoma or cholangiocarcinoma. 25.The method of claim 14, wherein the second therapeutic agent is selectedfrom decitabine, gemcitabine, cisplatin, carboplatin, oxaliplatin,adriamycin, liposomal doxorubicin, taxol, docetaxel, trametinib,binimetinib, cobimetinib, durvalumab, atezolizumab, sintilimab,toripalimab, camrelizumab, tislelizumab, nivolumab, or pembrolizumab.26. The method of claim 25, wherein the second therapeutic agent isselected from docetaxel, liposomal doxorubicin, cobimetinib,pembrolizumab, or decitabine.
 27. The method of claim 26, wherein thesecond therapeutic agent is cobimetinib.